Although the overall burden of tuberculosis (TB) in the WHO European region is declining, TB remains a major health issue worldwide. TB is among the ten most deadly diseases in low income countries, with 1.5 million TB deaths in 2020 (up from 1.4 million in 2019).^[1]TB incidence in Bulgaria is above the EU average with 1344 new diagnosis per year (19.2‱).^[2] While a quarter of the world’s population is infected with M. tuberculosis (МТВ), only 10% of them will ever develop the disease. However, activation of latent TB infection (LTBI) is potentiated in the settings of immune deficiency with different origin.^[3] Timely diagnosis and identification of cases with high risk of progression and activation are needed to limit TB spread, while avoiding heavy prophylaxis and treatment protocols, and selection of multi-resistant MTB strains.

Adaptive T-cell response is critical in MTB infection, with a leading role of Th1 effectors. Consequently, interferon gamma-release assays (IGRA) based on detection of memory MTB-specific T cells have gained wide diagnostic application.^[4–6] Yet, it has become clear that they can neither predict the course of infection nor the need of specific therapy. Several studies have proposed that protective T cell response is associated with a multicytokine profile of effector cells (IL-2, IFN-γ, TNF-α) which activate macrophages for phagocytosis and bacteriolysis.^[7,8]

The type of the immune response specific to MTB may actually depend on a wide range of factors associated with both innate and adaptive mechanisms. Recent data points out several effector and regulatory cytokines with possible role in the pathogenesis of MTB infection. A balanced immune inflammation is crucial for the induction of an adaptive immune response and efficient macrophage activation. IL-17 plays a key role in early neutrophil-mediated pulmonary inflammatory responses, T cell-mediated IFN-γ production and granuloma formation in the lung.‌^[9,10] Accumulation of Treg in chronic infections limits inflammation-associated pathology but may also affect pathogen-specific response through soluble mediators (IL-10, TGF-β) or direct cell contact.^[11]

Eicosanoids are a family of bioactive lipid mediators resulting from the metabolism of arachidonic acid that, similarly to cytokines, participate in inflammation and play a key role in shaping the adaptive response to MTB. Prostaglandins are the initial mediators of inflammatory response and stimulate recruitment of neutrophils and monocytes while lipoxins have anti-inflammatory activities and contribute to the resolution phase of the immune response.‌^[12] The balance between pro- and anti-inflammatory eicosanoids may shape the course of infection.^[13–15] Disease severity in TB has been associated with reduced ratio of prostaglandin E2 (PGE2)/lipoxin A4 (LXA4), rather than changes in the absolute levels of specific metabolites. A key counter-regulation between protective IL-1α, PGE2, and IL-17 signals against the pro-bacterial effects of IFNαβ/LXA4/IL-10 has been demonstrated in mice but awaits further study in men. It remains unclear whether eicosanoids can serve as biomarkers to distinguish activation of latent TB, to evaluate response to specific therapy or predict the outcome of a recent contact.^[16–18]

In the present study, we evaluated the dynamics of cytokine secretion in association with the lipid mediators PGE2 and LXA4 at different stages of MTB infection: after a recent contact, in established latent phase, and in active untreated, and treated disease.

The need for biomarkers predicting the course of MTB infection, as well as the necessity of specific therapy, is well recognized. Based on the experience with IGRA assays, we and others have concluded that bulk MTB-specific interferon-gamma responses of CD4 and CD8 T cells do not differentiate between MTB stages.^[19] A systematic review of the MTB-specific responses of 100 cytokines concluded that the most frequently studied cytokines were IFN-γ, IL-2, TNF-α, IP-10, IL-10, and IL-13 or combinations of them, rendering heterogenous findings and underlining the need for further well-designed studies.^[20]

We studied the induced expression of IFN-γ, TNF-α, IL-2, IL-17, IL-6, IL-10 and the lipid mediators PGE2 and LXA4, aiming to discriminate between QFT(-) RC, LTBI, and ATB and, possibly, to predict the course of MTB infection. Our results showed that IL-17 was the best discriminating cytokine differing significantly between each of the studied groups. In addition to high IL-17 expression, RC were differentiated by a high potential for TNF-α, IFN-γ, and IL-2 secretion. In contrast, ATB donors have partially preserved only the ability to produce TNF-α. Importantly, in RC stimulated IL-6 level was significantly lower than in the other groups pointing to preserved regulatory mechanisms at that early point of possible infection.

The key role of IL-17 in the course of TB infection has been related to the early neutrophil-mediated pulmonary inflammatory responses, T cell-mediated IFN-γ production, and granuloma formation in the lung.^[9,10] On the other hand, recent data from clinical trials and post-marketing surveillance of biological therapy suggested that IL-17 cells may be dispensable for LTBI control.^[21] Our results confirm the exhaustion of Th17 effectors at later stages of MTB infection, in line with a previously demonstrated increase of the Th17-specific inhibitory Treg subset in ATB donors.^[19]

The critical role of TNF-α in both early and latent infection has been demonstrated by neutralization of TNF and TNFR in mice, resulting in severe inflammation, uncontrolled infection or reactivation of latent TB infection.‌^[22–24] TNF-α triggers MTB killing by activating phagocytosis in macrophages, promotes dendritic cells maturation, and is responsible for the formation and maintenance of granulomas.^[25] While most studies have explored TNF-α expression in ATB and LTBI, a recently published study by Reichler et al. proposed an association of higher baseline TNF-α values with later development of incident TB among contacts.^[26]

The precise role of PGE2 and LXA4 in the development of adaptive immunity during human TB remains uncertain. A beneficial role of PGE2 is supported by the facts that PGE synthase-deficient mice and mice lacking the PGE2 receptor EP2 have increased susceptibility to MTB infection.^[27,28] On the other hand, high concentrations of PGE2 suppress T cell-mediated immunity against MTB and contribute to the expansion of regulatory T cells.^[29]

Lipoxin A4 (LXA4) also modulates innate and adaptive immune responses by exerting either pro-inflammatory or pro-resolution effects.^[29–31] Ligation of the lipoxin A4 (LXA4) receptor on neutrophils arrests their migration, but LXA4 is also the dominant lipid mediator produced by macrophages infected with virulent MTB and responsible for induction of necrosis. LXA4 blocks the synthesis of prostaglandin Е2 (PGE2), the latter being critical to avoid necrosis. Most probably, the issue of infection is determined by overlapping regulatory networks that function in coordination or antagonism.^[15,32]

In our hands, PGE2 and LXA4 did not differ significantly between the studied groups, while their ratio clearly discriminated between situation with high bacterial load as untreated ATB, and contained infection (LTBI, ATB-T). Increased PGE2/LXA4 ratio in the settings of ATB did not result from isolated increase of PGE2 or decrease of LXA4 but reflected changes in both mediators, confirming the idea about balanced regulation of proinflammatory and protective lipid mediators. This balance is close to one in the absence of immune activation (healthy controls, LTBI, contained early infection in RC), and is perturbed in the presence of bacterial load and/or inadequate regulation.

Few studies in humans have measured eicosanoid mediators during the stages of TB infection, with somewhat contradictory results. Thus, unlike us, Nore et al. reported increased LXA4 in untreated ATB compared to LTBI, while levels of PGE2 showed no difference between clinical stages of MTB infection and were not affected upon treatment.^[18] However, they measured native plasma concentrations and not the stimulated ones. On the other hand, higher levels of total monocytes were observed in ATB, with markedly increased expression of PGE2 and LXA4-specific enzymes upon PPD stimulation. This second model is closer to our stimulation-induced profiles, and the levels of PGE2 and LXA4 detected in our study may reflect induced monocyte production.

In line with us, Kumar et al. described significantly increased plasma levels of LXA4, and PGE2 in TB as compared to healthy controls, as well as significantly increased levels of LXA4 in TB individuals with bilateral or cavitary disease and a higher bacterial burden, while anti-tuberculosis therapy diminished the levels of LXA4 and PGE2.^[16] Pellegrini et al. recently showed that PGE2 potently suppresses the MTB-specific immune response by reducing the expression of co-stimulatory receptors (CD80, PDL-1, MHC-II), lymphocyte proliferation, and production of IFN-γ and TNF-α.^[33] These results suggest that PGE2 might be attenuating the excessive inflammatory immune response caused by MTB. These findings corroborate with our results showing an increased PGE2/LXA4 ratio in case of active MTB infection (infected RC and ATB). On the other hand, it was shown that a disrupted regulatory interaction between IL-10 and PGE2 leads to excessive expression of PGE2 and impaired killing of intracellular bacteria. ^[33] That might be the case in ATB where extremely high PGE2/LXA4 ratio was associated with a significantly decreased IL-10 potential (Fig. 3B).

The strong points of our study are the combined evaluation of cytokines and eicosanoid mediators in well-defined groups corresponding to different stages of MTB infection. In addition, we provided data on PGE2/LXA4 ratio in healthy controls. The major limitations in this study are the relatively small size of studied groups, and the cross-sectional design of the study. Further prospective studies following-up recent contacts and reactivated LTB are needed to assess the predictive value of PGE2/LXA4.

In conclusion, we propose stimulated PGE2/LXA4 ratio as a biomarker sensing the balance between physiological activation that promotes adaptive immunity, and suppression of excessive pathological inflammation. This balance indicates early control of recent infection, and containment of chronic infection in a latent state. A significant increase of PGE2/LXA4 ratio coupled with high IL-10 level signals instauration of infection after a recent contact, while the increased ratio and low TNF-α level point to inefficient granuloma formation in the settings of ATB.

M.N. designed the study, analyzed data, and drafted the manuscript; Y.T. performed the evaluation of PGE2, LXA4, cytokines, and analyzed data; R.E. performed the evaluation of PGE2, LXA4, cytokines, and analyzed data; V.M. enrolled volunteers, and collected data, E.B., Y.A., and A.B. performed microscopic examinations of AFB, culture examination by liquid (BACTEC - MGIT) and solid (Lowenstein-Jensen) media, and DST by LPA (MBTDR plus).